sRAGE mimetibody, compositions, methods and uses

ABSTRACT

Mammalian sRAGE mimetibody polypeptides and nucleic acids are disclosed. Methods of utilizing the polypeptides to reduce or inhibit the binding of RAGE and its ligands and to treat RAGE-related diseases are also disclosed.

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application Ser.No. 60/613,247, filed 27 Sep. 2004, the entire contents of which isincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to mammalian sRAGE mimetibodies and theiruse as therapeutics.

BACKGROUND OF THE INVENTION

The Receptor for Advanced Glycated Endproducts (RAGE) is a member of theimmunoglobulin superfamily of cell-surface molecules.

It was originally identified and characterized as a cellular receptorfor glucose (aldose sugar)-modified proteins or Advanced GlycatedEndproducts (AGE) (Schmidt et al., J. Biol. Chem. 267: 14987-14997,(1992); Neeper et al., J. Biol. Chem. 267: 14998-15004, (1992)). AGEhave been implicated in a variety of disorders associated with diabetesand aging (reviewed in Schmidt et al., Nature Med. 1: 1002-1004,(1995)). Binding of AGE to RAGE induces inflammatory responses in bloodvessel walls, which can trigger or aggravate the pathogenesis of macro-or micro-angiopathy.

RAGE has also been reported to interact with other ligands, includingamphoterin, a matrix-associated polypeptide that promotes outgrowth ofcultured cortical neurons derived from developing brain (Hori et al., J.Biol. Chem. 270: 25752-25761, (1995)). Amphoterin, also known in theliterature as high mobility group B1 (HMGB1), has been shown tocontribute to a variety of disease states including cancer, inflammatoryconditions and sepsis. See Lotze and DeMarco, Curr. Opin. Investig.Drugs 4: 1405-1409 (2003); Scaffidi et al., Nature 418: 191-195 (2002);and Wang et al., Science 285: 248-251 (1999). The expression of RAGE ismarkedly enhanced and co-localizes with that of amphoterin at theleading edge of advancing neurites, which indicates a potentialcontribution to cellular migration and in pathologies such as tumorinvasion. It has been reported that either anti-RAGE F(ab′)₂ or solubleRAGE inhibited neurite outgrowth on amphoterin-coated matrices (Taguchiet al., Nature 405: 354-360, (2000)). Soluble RAGE (sRAGE) is theextracellular domain of the receptor. In addition, blockade ofRAGE-amphoterin binding decreased growth and metastases of bothimplanted tumors and tumors developing spontaneously in susceptiblemice, Id.

RAGE has also been identified as a receptor on neurons and microglia forβ-amyloid, a polypeptide linked to the pathogenesis of neuronal toxicityand death in Alzheimer's disease (Yan et al., Nature 382: 685-691(1996)). Expression of RAGE is particularly increased in neurons closeto deposits of β-amyloid peptide and to neurofibrillary tangles. Inmice, RAGE mediates the transport of human β-amyloid-40 and -42 acrossthe blood-brain barrier (Deane et al., Nature Med. 9: 907-913 (2003)).Inhibition of the RAGE-ligand interaction, either by anti-RAGE IgG orsoluble RAGE, neither of which is transported across the blood-brainbarrier, suppressed the accumulation of β-amyloid in brain parenchyma ina mouse model of Alzheimer's disease, Id.

RAGE is also a central cell surface receptor for S100A2 and relatedmembers of the S100/calgranulins superfamily (Hofmann et al., Cell 97:889-901 (1999)). Interaction of S100A2 and cellular RAGE on endothelium,mononuclear phagocytes and lymphocytes triggers cellular activation andgeneration of key proinflammatory mediators. In murine models, blockadeof S100A2/RAGE quenched delayed-type hypersensitivity and inflammatorycolitis by arresting activation of central signaling pathways andexpression of inflammatory gene mediators, Id.

Studies have also shown that binding of RAGE by a ligand triggersactivation of key cell signaling pathways, such as p21(ras), MAPkinases, NF-κb, and cdc42/rac thereby reprogramming cellular properties.For example, upon ligand binding, RAGE initiates a signaling cascadethat results in the translocation of NF-κb to the nucleus and increasedexpression of adhesion molecules, procoagulant molecules andinflammatory proteins (Kislinger et al., J. Biol. Chem. 274: 31740-31749(1999)).

Studies have indicated that RAGE interacts with various moleculesimplicated in homeostasis, development, inflammation, and certaindiseases such as Type 2 diabetes and Alzheimer's disease. Accordingly,it would generally be desirable to block or otherwise inhibit theseinteractions, when associated with a disease state, to treat or preventthe associated pathologies.

sRAGE has been shown to be efficacious in animal disease models foratherosclerosis, tumor growth and metastasis, colitis, delayed-typehypersensitivity, experimental allergic encephalomyelitis, andAlzheimers disease (Park et al., Nature Med. 4: 1025-1031 (1998);Taguchi et al., supra; Hofmann et al., supra; Yan et al., supra) and canbe purified or expressed recombinantly. sRAGE lacks the transmembraneand extracellular domains of full-length RAGE and has threeimmunoglobulin-like regions: an N-terminal region most similar to animmunoglobulin variable domain (V domain) followed by two regionsresembling immunoglobulin constant regions (C domains).

SRAGE has been used extensively in vitro and in vivo to studyRAGE-ligand interactions. However, the half-life of sRAGE in rats is 24hours and therefore sRAGE itself is impractical as a therapeutic fordisease. Further, an anti-RAGE IgG would have to bind and inhibit largeamounts of RAGE normally expressed in tissues such as lung and mayinduce death of normal cells. Thus, a need exists for a modified sRAGEthat will overcome the short half-life while retaining the AGE bindingfunction of RAGE.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A to 1E show sRAGE mimetibody binding to AGEs. The mimetibodiestested include 1A: V in IgG1 with C220A and L234A/L235A mutations(V-G1)(SEQ ID NO: 5); 1B: V—C—C in IgG1 with C220A and L234A/L235Amutations (VCC-G1)(SEQ ID NO: 3); 1C: V in IgG4 with S228P and P234A,L235A mutations (V-G4)(SEQ ID NO: 13); 1D: V—C in IgG4 with S228P andP234A/L235A mutations (VC-G4)(SEQ ID NO: 11); and 1E: V—C—C in IgG4 withS228P and P234A/L235A mutations (VCC-G4)(SEQ ID NO: 9).

FIG. 2 shows the reduction of AGE binding to U937 cells by sRAGEmimetibodies.

FIGS. 3A to 3E show sRAGE mimetibody inhibition of AGE-induced mRNAexpression for tissue factor (FIG. 3A); VEGF (FIG. 3B); E-selectin (FIG.3C); RAGE (FIG. 3D); and IL-6 (FIG. 3E).

SUMMARY OF THE INVENTION

One aspect of the invention is a polypeptide having the generic formula(II):(Rg-Lk-V2—Hg—C_(H)2—C_(H)3) (t)  (II)where Rg is a mammalian sRAGE sequence, Lk is a polypeptide or chemicallinkage, V2 is a portion of a C-terminus of an immunoglobulin variableregion, Hg is at least a portion of an immunoglobulin variable hingeregion, C_(H)2 is an immunoglobulin heavy chain C_(H)2 constant regionand C_(H)3 is an immunoglobulin heavy chain C_(H)3 constant region and tis independently an integer from 1 to 10.

Another aspect of the invention is a polypeptide comprising apolypeptide having the sequence shown in SEQ ID NO: 3, 5, 7, 9, 11 or13.

Another aspect of the invention is a polynucleotide comprising apolynucleotide having the sequence shown in SEQ ID NO: 4, 6, 8, 10, 12or 14 or a complementary sequence.

Another aspect of the invention is a polynucleotide comprising apolynucleotide encoding the amino acid sequence shown in SEQ ID NO: 3,5, 7, 9, 11 or 13.

Another aspect of the invention is a method of modifying the biologicalactivity of RAGE in a mammal comprising administering an sRAGEmimetibody composition to the mammal.

Another aspect of the invention is a method of reducing the symptoms of,or treating at least one RAGE-related condition or disorder, comprisingadministering an sRAGE mimetibody composition to a patient in needthereof.

DETAILED DESCRIPTION OF THE INVENTION

All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as though fully set forth. Single letter amino acid codes areused herein as understood by those skilled in the art. Numbering ofamino acid residues in immunoglobulin constant regions is based onresidue one being the N-terminal amino acid in a wild type IgG1 or IgG4Fc domain.

The present invention provides polypeptides having the properties andactivities of mammalian sRAGE wherein the polypeptides also mimicdifferent types of immunoglobulin molecules such as IgA, IgD, IgE, IgG,or IgM, and any subclass thereof, such as IgA1, IgA2, IgG1, IgG2, IgG3or IgG4, or combinations thereof, hereinafter referred to as “sRAGEmimetibodies.” The invention also provides nucleic acids encoding sRAGEmimetibodies, vectors containing these nucleic acids, host cells,compositions and methods of making and using sRAGE mimetibodies.

Mimetibody Polypeptides and Compositions

The present invention generally relates to mimetibody polypeptideshaving the generic formula (I):(Pep-Lk-V2-Hg—C_(H)2—C_(H)3) (t)  (I)

where Pep is a polypeptide having a desired biological property, Lk is apolypeptide or chemical linkage, V2 is a portion of a C-terminus of animmunoglobulin variable region, Hg is at least a portion of animmunoglobulin hinge region, C_(H)2 is an immunoglobulin heavy chainC_(H)2 constant region and C_(H)3 is an immunoglobulin heavy chainC_(H)3 constant region and t is independently an integer of 1 to 10.

More particularly, the present invention relates to sRAGE mimetibodypolypeptides that are capable of blocking or reducing interactionsbetween RAGE and its respective ligands such as AGE, amphoterin,S100/calgranulin and β-amyloid. The polypeptides have the genericformula (II):(Rg-Lk-V2—Hg—C_(H)2—C_(H)3) (t)  (II)

where Rg is a mammalian sRAGE sequence, Lk is a polypeptide or chemicallinkage, V2 is a portion of a C-terminus of an immunoglobulin variableregion, Hg is at least a portion of an immunoglobulin hinge region,C_(H)2 is an immunoglobulin heavy chain C_(H)2 constant region andC_(H)3 is an immunoglobulin heavy chain C_(H)3 constant region and t isindependently an integer of 1 to 10.

As used herein, “soluble RAGE” or “sRAGE” encompasses RAGE-derivedpolypeptides that lack the transmembrane and intracellular regionsassociated with full-length RAGE. An exemplary mammalian sRAGEpolypeptide is the extracellular domain of human sRAGE having the aminoacid sequence shown in SEQ ID NO: 1. It is readily understood by thoseskilled in the art that sRAGE polypeptides can also include biologicallyactive fragments of SEQ ID NO: 1, amino acid sequences that aresubstantially homologous to SEQ ID NO: 1 as well as polypeptides thatcan mimic the binding activity of sRAGE. It will also be understood bythose skilled in the art that the mammalian sRAGE polypeptides useful inthe present invention can include a native N-terminal leader sequence(amino acids 1-30 of SEQ ID NO: 1) or other leader sequence tofacilitate excretion of the polypeptides of the invention from theirproduction cell line.

The term “biologically active fragment” as used herein, refers toportions of sRAGE polypeptides that can specifically bind to an sRAGEligand. Examples of biologically active fragments of sRAGE include, butare not limited to, one or more of the Ig-like domains, such as thesingle V domain (amino acids 31-106 of SEQ ID NO: 1), the V domain and aC domain (amino acids 31-215 of SEQ ID NO: 1), or the V domain and bothC domains (amino acids 31-308 of SEQ ID NO: 1).

The term “substantially homologous” in the context of polypeptides,refers to two amino acid sequences which, when optimally aligned, are atleast about 80% homologous or at least about 85% homoglous or at leastabout 90% homoglous or at least about 95% homologous. Alignment forpurposes of determining percent amino acid homology can be achieved invarious ways that are within the skill in the art, for example, by usingthe default settings for the AlignX component of Vector NTI Suite 8.0(Informax, Frederick, Md.) or publicly available computer software suchas BLAST (Altschul et al., J. Mol. Biol. 215: 403-410 (1990)). Apolypeptide sequence substantially homologous to SEQ ID NO: 1 has atleast one amino acid substitution, deletion or insertion.

In the polypeptides of the invention, the linker portion (Lk) providesstructural flexibility by allowing the mimetibody to have alternativeorientations and binding properties. Exemplary linkers includenon-peptide chemical linkages or one to 20 amino acids linked by peptidebonds, wherein the amino acids are selected from the 20 naturallyoccurring amino acids. The linker portion can include a majority ofamino acids that are sterically unhindered, such as glycine, alanine andserine and include GS, poly GS, GGGS (SEQ ID NO: 32), polymers of GGGSand GSGGGS (SEQ ID NO: 33) or any combination thereof. Other exemplarylinkers within the scope of the invention may be longer than 20 residuesand may include residues other than glycine, alanine and serine.

In the polypeptides of the invention, V2 is a portion of a C-terminaldomain of an immunoglobulin variable region such as a heavy chainvariable region. An exemplary V2 amino acid sequence is GTLVTVSS (SEQ IDNO: 16).

In the polypeptides of the invention, Hg is a portion of the hingedomain of an immunoglobulin variable region such as a heavy chainvariable region. Exemplary H amino acid sequences includeEPKSCDKTHTCPPCP (SEQ ID NO: 17), EPKSADKTHTCPPCP (SEQ ID NO: 18),ESKYGPPCPSCP (SEQ ID NO: 19), ESKYGPPCPPCP (SEQ ID NO: 20) and CPPCP(SEQ ID NO: 21).

In the polypeptides of the invention, C_(H)2 is an immunoglobulin heavychain C_(H)2 constant region. Exemplary C_(H)2 amino acid sequencesinclude: (SEQ ID NO: 22)APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAK, (SEQ IDNO: 23) APEAAGGPSVFLFPPKPKDTLNISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAK, (SEQ IDNO: 25) APEFLGGPSVFLFPPKPKDTLNISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPS SIEKTISKAK and (SEQID NO: 26) APEAAGGPSVFLFPPKPKDTLNISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPS SIEKTISKAK.

In the polypeptides of the invention, C_(H)3 is an immunoglobulin heavychain C_(H)3 constant region. Exemplary C_(H)3 amino acid sequencesinclude: (SEQ ID NO: 24)GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK and (SEQ IDNO: 27) GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKS LSLSLGK.It will be recognized by those skilled in the art that the C_(H)3 regionof the polypeptides of the invention may have its C-terminal amino acidcleaved off when expressed in certain recombinant systems.

In the polypeptides of the invention, the FcRn scavenger receptorbinding site of the immunoglobulin molecules is preserved at thejunction of the C_(H)2 and C_(H)3 region. Since FcRn binding enables thereturn of pinocytosed immunoglobulin back to the extracellular space, itis expected that the half-life of sRAGE mimetibodies will besignificantly extended relative to sRAGE.

The polypeptides of the invention can optionally comprise, at theirN-terminal, a portion of an N-terminus of an immunoglobulin variableregion, designated V1 as shown in Formula III. Exemplary V1 amino acidsequences include QIQ or QVQ.(V1-Rg-Lk-V2—Hg—C_(H)2—C_(H)3) (t)  (III)

In one embodiment of the polypeptides of the invention, the monomericstructure (Rg-Lk-V2—Hg—C_(H)2—C_(H)3) can be linked to other monomersnon-covalently or by covalent linkage, such as, but not limited to, aCys-Cys disulfide bond. It is thought that this structure will allowstable dimerization of sRAGE and increase its affinity to RAGE ligands.

Another embodiment of the present invention is a polypeptide comprisinga polypeptide according to formula (II) where Rg is a single copy of theRAGE full-length extracellular domain (SEQ ID NO: 1), V2 is a J regionof a naturally occurring IgG (SEQ ID NO: 16), Hg is a complete IgG1hinge region with a Cys220Ala (C220A) substitution (SEQ ID NO: 18) andC_(H)2 and C_(H)3 are of the IgG1 isotype subclass with Leu234Ala andLeu235Ala substitutions (L234A/L235A) (SEQ ID NO: 28). The completepolypeptide sequence of this embodiment is shown in SEQ ID NO: 3.

Another embodiment of the present invention is a polypeptide comprisinga polypeptide according to formula (II) where Rg is a single copy of theV domain of the RAGE full-length extracellular domain (residues 31 to106 of SEQ ID NO: 1), V2 is a J region of a naturally occurring IgG (SEQID NO: 16), Hg is a complete IgG1 hinge region with a C220A substitution(SEQ ID NO: 18) and C_(H)2 and C_(H)3 are of the IgG1 isotype subclasswith L234A/L235A substitutions (SEQ ID NO: 28). The complete polypeptidesequence of this embodiment is shown in SEQ ID NO: 5.

Another embodiment of the present invention is a polypeptide comprisinga polypeptide according to formula (II) where Rg is a single copy of theRAGE full-length extracellular domain (SEQ ID NO: 1), V2 is a J regionof a naturally occurring IgG (SEQ ID NO: 16), Hg is the complete IgG4hinge region (SEQ ID NO: 19) and C_(H)2 and C_(H)3 are of the IgG4isotype subclass (SEQ ID NO: 29). The complete polypeptide sequence ofthis embodiment is shown in SEQ ID NO: 7.

IgG1 and IgG4 subclasses differ in the number of cysteines in the hingeregion. Like the IgG1 subclass, there are two cysteines in the IgG4hinge that participate in the disulfide bonding between heavy chains.However, the cysteine in IgG1 hinge that is normally involved indisulfide bonding to the light chain is absent in the IgG4 hinge.Therefore, the IgG4 hinge is less flexible than the IgG1 hinge.

In addition, the two isotypes differ in their ability to mediatecomplement dependent cytotoxicity (CDC) and antibody-dependent cellularcytotoxicity (ADCC). CDC is the lysing of a target in the presence ofcomplement. The complement activation pathway is initiated by thebinding of the first component of the complement system (C1q) to amolecule complexed with a cognate antigen. IgG1 is a strong inducer ofthe complement cascade and subsequent CDC activity, while IgG4 haslittle complement-inducing activity.

ADCC is a cell-mediated reaction in which nonspecific cytotoxic cellsthat express Fc receptors (FcRs) (e.g., Natural Killer (NK) cells,neutrophils, and macrophages) recognize bound antibody on a target celland subsequently cause lysis of the target cell. The IgG1 subclass bindswith high affinity to the Fc receptor and contributes to ADCC while IgG4binds only weakly. The relative inability of IgG4 to activate effectorfunctions is desirable since delivery of the mimetibody to cells withoutcell killing is possible.

Furthermore, the binding site for the FcRn scavenger receptor is presentin IgG4 and IgG1 isotypes and both have similar binding characteristics.Therefore, the pharmacokinetics of the IgG1 and IgG4 mimetibodies of theinvention are expected to be similar.

The hinge-C_(H)2—C_(H)3 portion of the immunoglobulin region(Hg—C_(H)2—C_(H)3) may also be extensively modified to form variants inaccordance with the invention. For example, one or more native sitesthat provide structural features or functional activity not required bythe mimetibody molecules could be removed. These sites may be removedby, for example, substituting or deleting residues, inserting residuesinto the site, or truncating portions containing the site. ExemplaryHg—C_(H)2—C_(H)3 variants are discussed below.

1. Sites involved in disulfide bond formation can be removed by deletionor substitution with other amino acids in the mimetibodies of theinvention. Typically, the cysteine residues present in these motifs areremoved or substituted. Removal of these sites may avoid disulfidebonding with other cysteine-containing proteins present in themimetibody-producing host cell or intra-heavy chain disulfide bonding inIgG4-based constructs while still allowing for a dimericC_(H)3—C_(H)2-hinge domain that is held together non-covalently.

Most IgG type antibodies, such as IgG1, are homodimeric molecules madeup of two identical heavy (H) chains and two identical light (L) chains,typically abbreviated H₂L₂. Thus, these molecules are generally bivalentwith respect to antigen binding, i.e., both antigen binding (Fab) armsof the IgG molecule have identical binding specificity.

IgG4 isotype heavy chains contain a CPSC (SEQ ID NO: 15) motif in theirhinge regions capable of forming either inter- or intra-heavy chaindisulfide bonds, i.e., the two Cys residues in the CPSC motif maydisulfide bond with the corresponding Cys residues in the other H chain(inter) or the two Cys residues within a given CPSC motif may disulfidebond with each other (intra). It is believed that in vivo isomeraseenzymes are capable of converting inter-heavy chain bonds of IgG4molecules to intra-heavy chain bonds and vice versa (Aalberse andSchuurman, Immunology 105, 9-19 (2002)).

Accordingly, since the HL pairs in those IgG4 molecules with intra-heavychain bonds in the hinge region are not covalently associated with eachother, they may dissociate into HL monomers that then reassociate withHL monomers derived from other IgG4 molecules forming bispecific,heterodimeric IgG4 molecules. In a bispecific IgG antibody the two Fabsof the antibody molecule differ in the epitopes that they bind.Substituting Ser228 in the hinge region of IgG4 with Pro results in“IgG1-like behavior,” i.e., the molecules form stable disulfide bondsbetween heavy chains and therefore, are not susceptible to HL exchangewith other IgG4 molecules.

2. The H—C_(H)2—C_(H)3 can be modified to make the mimetibodies of theinvention more compatible with a selected host cell. For example, when amimetibody of the invention is expressed recombinantly in a bacterialcell such as E. coli, the Pro-Ala sequence in the hinge may be removedto prevent digestion by the E coli enzyme proline iminopeptidase.

3. A portion of the hinge region can be deleted or substituted withother amino acids in the mimetibodies of the invention to preventheterogeneity in the products expressed in a selected host cell.

4. One or more glycosylation sites can be removed in the mimetibodies ofthe invention. Residues that are typically glycosylated (e.g., Asn) mayconfer an Fc-dependent, cell-mediated cytolytic activity to themimetibody. Such residues may be deleted or substituted with residuesthat are not glycosylated such as Ala.

5. Sites involved in interaction with complement, such as the C1qbinding site, are removed in the mimetibodies of the invention.

6. Sites can be removed that affect binding to Fc receptors other than aFcRn salvage receptor in the mimetibodies of the invention. For example,the Fc receptors involved in ADCC activity can be removed in themimetibodies of the invention. For example, mutation of Leu234/Leu235 inthe hinge region of IgG1 to L234A/L235A or Phe234/Leu235 in the hingeregion of IgG4 to P234A/L235A minimizes FcR binding and reduces theability of the immunoglobulin to mediate complement dependentcytotoxicity and ADCC.

One embodiment of the present invention is an sRAGE mimetibody accordingto formula (II) where the Hg—C_(H)2—C_(H)3 is from the IgG4 subclass(SEQ ID NO: 30) and contains a Ser228Pro (S228P) substitution andP234A/L235A mutations (SEQ ID NO: 31). The complete polypeptidesequences of exemplary sRAGE mimetibody polypeptides having thesemutations and the (V—C—C), (V—C) and (V) domains of sRAGE, are shownrespectively in SEQ ID NOs: 9, 11 and 13. These mimetibody constructsare expected be a homogeneous and stable population that does nottrigger FcR-mediated effector functions. The substitution and mutationsshown here are exemplary; Hg—C_(H)2—C_(H)3 domains within the scope ofthis invention may include other substitutions, mutations and/ordeletions.

As mentioned above, the amino acid sequences of specific examples ofmimetibodies of the invention are shown in SEQ ID NOs: 3, 5, 7, 9, 11and 13. The characterstics of these constructs are shown in Table 1(n.a.=not applicable). TABLE 1 Exemplary mimetibody constructs SEQ SRAGEIg L234A/ P234A/ ID NO: domains isotype C220A S228P L235A L235A 3 V-C-CIgG1 Yes n.a. Yes n.a. 5 V IgG1 Yes n.a. Yes n.a. 7 V-C-C IgG4 n.a. Non.a. No 9 V-C-C IgG4 n.a. Yes n.a. Yes 11 V-C IgG4 n.a. Yes n.a. Yes 13V IgG4 n.a. Yes n.a. Yes

The present invention includes sRAGE mimetibodies that are capable ofblocking or reducing interactions between RAGE and at least one of itsligands. Embodiments of the invention include sRAGE mimetibodies thatspecifically bind to AGEs, amphoterin, S100/calgranulin or β-amyloid orany combination thereof. The mimetibodies of the present invention canbind RAGE ligands with a wide range of affinities. Exemplary sRAGEmimetibodies bind at least one RAGE ligand with high affinity. Forexample, an sRAGE mimetibody can bind at least one of AGEs, amphoterin,S100/calgranulin, β-amyloid or any combination thereof with a Kd equalto or less than about 10⁻⁷M, 10⁻⁸, 10⁻⁹, 10⁻¹⁰, 10⁻¹¹ or 10⁻¹²M.

The affinity of an sRAGE mimetibody for a specific RAGE ligand can bedetermined experimentally using any suitable method, for example,methods using Biacore or KinExA instrumentation, ELISA and competitivebinding assays. sRAGE mimetibodies having specific ligand bindingcapabilities can be selected from libraries of sRAGE variants or sRAGEfragments by techniques known to those skilled in the art.

The sRAGE mimetibodies of the present invention are useful in treatingdisorders or symptoms resulting from abnormal receptor activation ofRAGE triggered by at least one ligand's binding. As describedpreviously, the association of RAGE and its ligands has been implicatedin a number of pathological conditions, such as but not limited to,Types 1 and 2 diabetes and Alzheimer's disease. Accordingly, anotheraspect of the present invention is pharmaceutical compositionscomprising at least one sRAGE mimetibody and a pharmaceuticallyacceptable carrier or diluent known in the art. The carrier or diluentcan be a solution, suspension, emulsion, colloid or powder.

An sRAGE mimetibody of the invention is formulated as a pharmaceuticalcomposition in a therapeutically or prophylactically effective amount.The term “effective amount” generally refers to the quantities ofmimetibody necessary for effective therapy, i.e., the partial orcomplete alleviation of the symptom or disorder for which treatment wassought. Included within the definition of effective therapy areprophylactic treatments intended to reduce the likelihood of onset ofthe above-described symptoms or disorders.

The composition can optionally comprise at least one further compound,protein or composition useful for treating the disease states discussedbelow. For example, combination with insulin, metformin, sulfonylureasor PPAR-γ agonists are contemplated in the treatment of Type 2 diabetes.Further, combination with anti-inflammatory agents to treat inflammatorydisorders and combination with chemotherapy agents to treat cancer arealso contemplated.

Nucleic Acids, Vectors and Cell Lines

Another aspect of the present invention is isolated nucleic acidmolecules comprising, complementary to or having significant identitywith a polynucleotide encoding at least one sRAGE mimetibody. Otheraspects of the present invention include recombinant vectors comprisingat least one isolated sRAGE mimetibody encoding nucleic acid moleculeand cell lines and organisms that are capable of expressing the nucleicacid molecules.

The nucleic acids, expression vectors and cell lines may generally beused to produce the mimetibody of the invention.

In one embodiment, the nucleic acid compositions of the invention encodepolypeptides having amino acid sequences identical to or substantiallyhomologous to any one of SEQ ID NOs: 3, 5, 7, 9, 11 or 13. Exemplarynucleic acid sequences that encode the polypeptide sequences shown inSEQ ID NOs: 3, 5, 7, 9, 11 and 13 are shown in SEQ ID NOs: 4, 6, 8, 10,12 and 14, respectively. Also provided are substantially similar nucleicacid sequences and allelic variations of the above-described nucleicacids.

The term “substantially similar” in the nucleic acid context, means thatthe segments, or their complementary strands, when properly aligned,with the appropriate nucleotide insertions or deletions, are identicalin at least 60% or at least about 70% or at least about 80% or at leastabout 90% or at least about 95-98% of the nucleotides. Values for %identity can be obtained from nucleotide sequence alignments generatedusing the default settings for the AlignX component of Vector NTI Suite8.0 (Informax, Frederick Md.).

Typically, the nucleic acids of the present invention are used inexpression vectors for the preparation of the sRAGE mimetibodypolypeptides of the invention. Vectors within the scope of the inventionprovide necessary elements for eukaryotic expression, including viralpromoter driven vectors, such as CMV promoter driven vectors, e.g.,pcDNA3.1, pCEP4 and their derivatives, Baculovirus expression vectors,Drosophila expression vectors and expression vectors that are driven bymammalian gene promoters, such as human Ig gene promoters. Otherexamples include prokaryotic expression vectors, such as T7 promoterdriven vectors, e.g., pET41, lactose promoter driven vectors andarabinose gene promoter driven vectors.

The present invention also relates to cell lines expressing sRAGEmimetibodies. The host cells can be prokaryotic or eukaryotic cells.Exemplary eukaryotic cells are mammalian cells, such as but not limitedto, COS-1, COS-7, HEK293, BHK21, CHO, BSC-1, HepG2, 653, SP2/0, NS0,293, HeLa, myeloma, lymphoma cells, or any derivative thereof. Mostpreferably, the host cells are HEK293, NS0, SP2/0 or CHO cells. The celllines of the present invention may stably express at least one sRAGEmimetibody. The cell lines may be generated by stable or transienttransfection procedures that are well known in the art.

The present invention further provides methods for expressing at leastone sRAGE mimetibody comprising culturing the cell lines underconditions wherein the sRAGE mimetibody is expressed in detectable orrecoverable amounts. The present invention also provides methods forgenerating at least one sRAGE mimetibody comprising translating thesRAGE mimetibody encoding nucleic acids under conditions in vitro or insitu, such that the sRAGE mimetibody is expressed in detectable orrecoverable amounts. The present invention also encompasses sRAGEmimetibodies produced by the above methods.

An sRAGE mimetibody can be recovered and purified by well-known methodsincluding, but not limited to, protein A purification, ammonium sulfateor ethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, hydroxylatpatite chromatographyand lectin chromatography. High performance liquid chroatography (HPLC)can also be employed for purification.

Methods of Use

The sRAGE mimetibodies are useful as, inter alia, research reagents andtherapeutic agents. In one aspect, the present invention relates to amethod of modifying the biological activities of RAGE comprisingproviding at least one sRAGE mimetibody to a mammal in need thereof. ThesRAGE mimetibody may decrease or inhibit RAGE-activated cell signalingcascades, such as but not limited to the NF-Kb pathway. The sRAGEmimetibody of the invention can thus diminish the increased expressionof adhesion molecules, procoagulant molecules and inflammatory proteinsdue to RAGE-initiated NF-Kb translocation into the nucleus. Inparticular, the sRAGE mimetibody may function as an antagonist of RAGE.The term “antagonist” is used in the broadest sense and includes amolecule that is capable of, directly or indirectly, partially or fullycounteracting, reducing or inhibiting one or more biological activitiesof RAGE. Examples of such biological activites of RAGE include itsbinding to AGEs, amphoterin, Sl00/calgranulin or □-amyloid, activationof NF-□b, p21(ras), MAP kinases or cdc42/rac as well as other activitiesknown to those skilled in the art.

The present invention further provides methods for reducing the symptomsof, or treating at least one RAGE-related condition or diseasecomprising administering a therapeutically effective amount of at leastone sRAGE mimetibody pharmaceutical composition to a patient in needthereof. As described above, such composition comprises an effectiveamount of at least one sRAGE mimetibody and a pharmaceuticallyacceptable carrier or diluent. The effective amount for a given therapy,whether curative or preventative, will generally depend upon maydifferent factors, including means of administration, target site andother medicants administered. Thus, treatment doses will need to betitrated to optimize safety and efficacy.

The conditions and diseases suitable for treatment using the methods ofthe present invention include but are not limited to, immune disorders,cardiovascular disorders, metabolic diseases, malignant disorders, andneurologic disorders. Non-limiting examples of these conditions ordisorders are Types 1 and 2 diabetes, Alzheimer's disease,atherosclerosis, tumor growth and metastasis, inflammation, colitis,delayed type hypersensititvity, multiple sclerosis and aging relateddisorders such as oxidant stress. These methods can optionally furthercomprise co-administration or combination therapies with any standardtherapy used to treat the diseases listed above.

The mode of administration can be any suitable route to deliver thepharmaceutically effective amount of sRAGE mimetibody of the presentinvention to a host. For example, the sRAGE mimetibody can be deliveredvia parenteral administration, such as subcutaneous, intramuscular,intradermal, intravenous or intranasal administration, or any othermeans known in the art.

The present invention is further described with reference to thefollowing examples. These examples are merely to illustrate aspects ofthe present invention and are not intended as limitations of thisinvention.

EXAMPLE 1 Cloning, Expression and Purification of an sRAGE mimetibody inMammalian Cells

Human sRAGE encoding cDNA was PCR amplified from human fetal lung5′-STRETCH Plus cDNA Library (Clontech, Palo Alto, Calif.). The firstround amplification was performed using forward primer5′-GTCCCTGGAAGGAAGCAGG-3′ (SEQ ID NO: 34) and reverse primer5′-TTTGGTACCCCTCAAGGCCCTCCAG-3′ (SEQ ID NO: 35).

A second round nested amplification was necessary to yield sufficientquantities of sRAGE cDNAs. The nested forward primers included an NcoIrestriction enzyme recognition site and the nested reverse primersincluded a KpnI site. Specifically, the V region was amplified usingforward primer 5′-TTTCCATGGCAGCCGGAACAGCAG-3′ (SEQ ID NO: 36) andreverse primer 5′-TTTGGTACCTCCATTCCTGTTCATTGCCTGG-3′ (SEQ ID NO: 37);and the V—C—C region was amplified using forward primer5′-TTTCCATGGCAGCCGGAACAGCAG-3′ (SEQ ID NO: 36) and reverse primer5′-TTTGGTACCGTGGCTGGAATGGGTGGCC-3′ (SEQ ID NO: 38).

The amplified PCR products (V region or V—C—C region of sRAGE) werecloned into the NcoI/KpnI site of an intermediate vector using standardcloning procedures. The EcoRI fragment of the assembled intermediateplasmids, containing promoter elements, enhancers and sRAGE encodingsequences were cloned into a mouse Ig gene promoter driven, human IgG1ΔCH1 Ala/Ala expression vector. Plasmids expressing a V regioncontaining sRAGE mimetibody and a V—C—C containing sRAGE mimetibody weregenerated.

The sRAGE IgG1 mimetibodies were stably expressed in mouse myeloma cellsSP2/0 and purified from the conditioned media using protein A affinitychromatography according to standard procedures.

The sRAGE mimetibodies were also cloned into an IgG4 Ser to Pro, Ala/Alascaffold. To amplify fragments encoding the V—C and the V—C—C sRAGEmimetibodies, the V—C—C containing plasmid was used as the template. Theforward primers included an NheI site, and the reverse primers includeda BamHI site. Specifically, the V—C region was amplified using forwardprimer 5′-TTTGCTAGCGCCACCATGGCAGCCGGAACAGCAGTT-3′ (SEQ ID NO: 39) andreverse primer 5′-TTTGGATCCGGGAAGGCCTGGGCTGAAGCTACA-3′ (SEQ ID NO: 40);and the the V—C—C region was amplified using forward primer5′-TTTGCTAGCGCCACCATGGCAGCCGGAACAGCAGTT-3′ (SEQ ID NO: 39) and reverseprimer 5′-TTTGGATCCGTGGCTGGAATGGGTGGCCACACA-3′ (SEQ ID NO: 41). The PCRproducts were digested using Nhe1 and BamH1. Because of an internalBamHI site in the V-like domain of RAGE, the PCR products were cleavedinto two fragments using NheI/BamHI: one with nucleotides 1-275 of SEQID NO: 2, the other with nucleotides 276-645 (V—C product) or 276-924(V—C—C product). Cloning of sRAGE mimetibody into the IgG4 expressionvectors involved two steps. First, the 1-275 fragment was cloned intothe NheI/BamHI site of human IgG4 ΔCH1, Ser to Pro, Ala/Ala expressionvector to generate an intermediate plasmid. The 276-645 or the 276-924fragment was subsequently cloned into the BamHI site of the intermediateplasmid to generate plasmid expressing V—C or V—C—C sRAGE mimetibody,respectively.

To clone the V-only region into the IgG4 Ser to Pro, Ala/Ala scaffold,PCR was performed using forward primer5′-TTTGCTAGCGCCACCATGGCAGCCGGAACAGCAGTT-3′ (SEQ ID NO: 39) and reverseprimer5′-TTTGGATCCTCCATTCCTGTTCATTGCCTGGCACCGGAAAATCCCCTCATCCTGAATCCCGACAGCCGGAAGGAA-3′ (SEQ ID NO: 42). The NheI/BamHI digested PCR product wascloned into the NheI/BamHI site of human IgG4 ΔCH1, Ser to Pro, Ala/Alaexpression vector in a single step.

The IgG4 mimetibodies were transiently expressed in HEK293E cells andpurified from the conditioned media using protein A affinitychromatography according to standard procedures.

EXAMPLE 2 Binding of AGEs to sRAGE Mimetibody

To prepare AGE, bovine serum albumin (BSA, Fraction V) (Sigma, St.Louis, Mo.) was incubated under non-reducing conditions with 50 mMglycoaldehyde in phosphate buffered saline (PBS) (pH 7.4) withoutcalcium chloride or magnesium chloride for 3 days at 37° C. To terminatethe reaction, the AGE were dialyzed with 10 volumes of PBS (pH 7.4). Thefinal protein concentration was determined using the Bicinchoninic Acidbased protein assay (Pierce, Rockford, Ill.). Samples were determined tocontain less than 1 endotoxin unit (EU) per microgram of protein usingLimulus amebocyte lysate (LAL) testing.

Solid phase ELISA was used to assess mimetibody binding to AGE. Briefly,96-well plates were coated overnight with 30 ug/ml AGE in 0.1 MCarbonate buffer, pH9.5 at 37° C. After washing in 0.15 M saline buffercontaining 0.02% (w/v) Tween-20, the wells were blocked with 1% (w/v)BSA, 0.05% Tween-20 in PBS for 2 hours at 37° C. After washing, plateswere incubated with varying concentrations of sRAGE mimetibody dilutedin PBS for 45 minutes at 37° C. Plates were washed and probed withHRP-labeled goat anti-human Fc (Jackson ImmunoResearch, West Groove,Pa.) diluted 1:1000 in 1% BSA/PBS for 20 minutes at 37° C. Followinganother washing step, plates were incubated for 15 minutes at roomtemperature with 100 μL/well of Sigma 104 Phosphate Substrate (Sigma).Substrate development was stopped by addition of 25 μL/well sulfuricacid (4N) and the absorbance was measured at 490 nm via an automatedplate spectrophotometer. As shown in FIG. 1, the mimetibodies tested alldemonstrated binding to AGE. The binding affinities (KD) are shown inTable 2. TABLE 2 Binding affinities (KD) of exemplary mimetibodyconstructs SRAGE KD SEQ ID NO: domains Ig isotype (μg/ml) 3 V-C-C IgG1,C220A, L234A/L235A 0.62 5 V IgG1, C220A, L234A/L235A 17.7 9 V-C-C IgG4,S228P, P234A/L235A 0.18 11 V-C IgG4, S228P, P234A/L235A 0.70 13 V IgG4,Ser −> Pro, Ala/Ala 0.89

EXAMPLE 3 SRAGE Mimetibody Reduces the Binding of AGE to RAGE

To measure the binding of the AGE to RAGE, a ¹²⁵-I binding assay wasperformed. Cells that express RAGE (U937 cells) were cultured in DMEMmedia with 10% FBS and 1% L-Glutamine, Na Pyruvate and non-essentialamino acids at 37° C., 5% CO₂, until 1.5×10⁶ confluence was reached.Cells were plated in 96-well filter plates at 150,000 cells/well andincubated with 30 ug/ml Fc control (IgG4 mimetibody backbone withoutsRAGE encoding sequence) for 30 minutes at 37° C. to block Fc receptors.AGE were labeled with ¹²⁵-I and the ¹²⁵-I-AGEs were added to cells intwo fold dilutions from 200 ug/ml to 0.1 ug/ml. After incubating at roomtemperature for 2 hours to allow maximal binding, the plates werefiltered and washed with PBS to retain cells on the nitrocellulosemembrane while allow unbound protein to pass through. The radioactivityon the membrane was measured using a Gamma counter. Non-specific bindingwas measured by co-incubation with 100+-fold excess of unlabeled AGE.The counts obtained fom the non-specific binding were subtracted fromthe total binding data to determine specific bidning. The affinity (KD)of AGE binding to U937 cells was calculated to be 11 ug/ml.

The ability of the sRAGE MMBs to reduce AGE binding to U937 cells wasdetermined by a competition assay. Briefly, ¹²⁵-I-AGE (11 ug/ml) wereincubated with sRAGE mimetibodies for 2 hours. The mimetibodies usedwere diluted two fold from 200 ug/ml to 0.1 ug/ml.

The mixture of ¹²⁵-I-AGE and sRAGE mimetibodies was added to U937 andthe binding of AGEs to U937 was measured using the binding assaydescribed above. As shown in FIG. 2, sRAGE mimetibodies testeddemonstrated reduction of AGE binding to the U937 cells. IC50 of thetested mimetibodies were calculated at approximately 90 nM.

EXAMPLE 4 SRAGE Mimetibody Reduces AGE-Induced Gene Expression

To show AGE stimulation of expression of certain genes, human umbilicalendothelial cells (HUVEC) were incubated with 30 ug/ml AGE, 600 ug/mlAGE, for 1, 4, 8 or 12 hours. As a positive control, cells wereincubated with 13 ug/ml Lipopolysaccharide (LPS), an inflammatorystimulant. As a negative control, cells were incubated with 600 ug/mlBSA. Cells were harvested and total RNA was isolated using RNeasy(Qiagen, Valencia, Calif.). The RNA was quantitated with the AgilentBioanalyzer. Quantitative PCR was performed using Assay-on-Demandprimers (Applied Biosystems, Foster City, Calif.) for E-Selectin(HS00174057), RAGE (HS00153957), VEGF (HS00173626), IL-6 (HS00174131),GAPDH (HS99999905) and Tissue Factor (HS00175225). The RT-PCR productswere quantitated relative to 18s RNA. As shown in FIG. 3, AGE, but notcontrol BSA, increased the mRNA level of each of the genes tested.

To demonstrate the ability of SRAGE mimetibodies to inhibitAGE-stimulated mRNA levels, HUVEC cells were incubated with 3.3 ug/mlsRAGE mimetibody in addition to 600 ug/ml AGE or 13.2 ug/ml sRAGEmimetibody in addition to 600 ug/ml AGE. RNA purification andquantitative PCR were performed as described above. As shown in FIG. 3,sRAGE mimetibodies inhibited the AGE-induction of the transcriptsencoding (3A) Tissue Factor, (3B) VEGF, (3C) E-Selectin, (3D) RAGE, and(3E) IL-6.

The present invention now being fully described, it will be apparent toone of ordinary skill in the art that many changes and modifications canbe made thereto without departing from the spirit or scope of theappended claims.

1. A polypeptide according to formula (II):(Rg-Lk-V2—Hg—C_(H)2—C_(H)3) (t)  (II) where Rg is a mammalian sRAGEsequence, Lk is a polypeptide or chemical linkage, V2 is a portion of aC-terminus of an immunoglobulin variable region, Hg is at least aportion of an immunoglobulin variable hinge region, C_(H)2 is animmunoglobulin heavy chain C_(H)2 constant region and C_(H)3 is animmunoglobulin heavy chain C_(H)3 constant region and t is independentlyan integer from 1 to
 10. 2. The polypeptide of claim 1 wherein Rg is abiologically active fragment of or a polypeptide substantiallyhomologous to SEQ ID NO:
 1. 3. The polypeptide of claim 1 wherein Rg hasthe amino acid sequence shown in residues 31 to 106, 31 to 215 or 31 to308 of SEQ ID NO:
 1. 4. The polypeptide of claim 1 wherein Hg, C_(H)2and C_(H)3 are of the IgG1 subclass.
 5. The polypeptide of claim 4wherein Cys220 of Hg is substituted with Ala, and Leu234 and Leu235 ofC_(H)2—C_(H)3 are mutated to Ala234 and Ala235.
 6. The polypeptide ofclaim 1 wherein Hg is of the IgG4 subclass, and C_(H)2 and C_(H)3 are ofthe IgG1 subclass.
 7. The sRAGE mimetibody polypeptide of claim 1wherein Hg, C_(H)2 and C_(H)3 are of the IgG4 subclass.
 8. Thepolypeptide of claim 1 wherein Ser228 of Hg is substituted with Pro andPhe234 and Leu235 of C_(H)2—C_(H)3 are mutated to Ala234 and Ala235. 9.The polypeptide of claim 1 wherein the polypeptide binds to at least oneRAGE ligand.
 10. The polypeptide of claim 9 wherein the ligand is atleast one of advanced glycated endproducts (AGE), amphoterin,S100/calgranulin and β-amyloid.
 11. A polypeptide comprising apolypeptide having the sequence shown in SEQ ID NO: 3, 5, 7, 9, 11 or13.
 12. A polynucleotide encoding a polypeptide according to claim 1.13. A polynucleotide comprising a polynucleotide having the sequenceshown in SEQ ID NO: 4, 6, 8, 10, 12 or 14 or a complementary sequence.14. A polynucleotide comprising a polynucleotide encoding the amino acidsequence shown in SEQ ID NO: 3, 5, 7, 9, 11 or
 13. 15. A vectorcomprising the polynucleotide of claim 13 or
 14. 16. A cell lineexpressing a polypeptide according to claim
 1. 17. A cell linecomprising the vector of claim
 15. 18. The cell line of claim 17 whereinthe cell line is HEK293, NSO, SP2/0 or CHO cells.
 19. A pharmaceuticalcomposition comprising an effective amount of at least one polypeptideaccording to claim 1 and a pharmaceutically acceptable carrier ordiluent.
 20. A method of modifying the biological activity of RAGE in amammal comprising administering the pharmaceutical composition of claim19 to the mammal.
 21. A method of reducing the symptoms of, or treatingat least one RAGE-related condition or disorder, comprisingadministering the pharmaceutical composition of claim 19 to a patient inneed thereof.
 22. The method of claim 21 wherein the RAGE-relatedcondition or disorder is an immune disorder, a cardiovascular disorder,a metabolic disease, a malignant disorder, or a neurological disorder.23. The method of claim 21 wherein the RAGE-related condition ordisorder is diabetes, Alzheimer's disease, atherosclerosis, tumor growthand metastasis, colitis, delayed type hypersensitivity, multiplesclerosis or oxidant stress.
 24. A method to produce a polypeptidecomprising the steps of culturing the cell line of claim 16 andpurifying the expressed polypeptide.